Nucleotides and nucleotide sugars are released in a regulated fashion from both excitable and non-excitable cells and act as extracellular signaling molecules to regulate a remarkably broad array of physiological responses. At least fifteen different P2 receptors recognize extracellular nucleotides as their cognate agonists; eight of these comprise a family of ubiquitously expressed metabotropic G protein-coupled receptors called the P2Y receptors. Signals emanating from these receptors convey important physiological and pathophysiological responses in essentially all tissues, and the potential for therapeutic interventions that target these processes is both very large and almost completely unrealized. The long-term objectives of this research are (i) to define the functional relationships between nucleotide release, extracellular metabolism, and P2Y receptor activation, (ii) to delineate the molecular details of the interaction of P2Y receptors with their agonists and downstream signaling proteins, and (iii) to develop high affinity receptor-selective agonists and antagonists for these receptors. In Specific Aim 1 we will continue to synthesize higher affinity P2Y1 receptor antagonists, to develop new high affinity radioligands for the P2Y1 receptor, to develop P2Y subtype-selective non-nucleotide agonists, and to develop new antagonists for the P2Y4 receptor and potentially other P2Y receptors. In Specific Aim 2 we will clone an uncharacterized member of the P2Y12 subfamily of P2Y receptors that exists in mammalian databases and determine its cognate agonist(s). Its downstream signaling specificities will be determined in a detailed set of experiments in which similar activities are defined for the other three members (P2Y 12, P2Y13, and P2Y14 receptors) of this subfamily. Although the molecular species of ectoenzymes that metabolize extracellular nucleotides have been identified, the functional relationship between these ectoenzymes, nucleotide release, and P2Y receptor activation is unknown. Therefore, in Specific Aim 3 we will study the ATP-activated P2Y2 receptor and ADP-activated P2Y1 receptor under the varying presence of NTPDase1, which metabolizes both ATP and ADP, or NTPDase2, which converts ATP to ADP. Natively expressed NTPDase and nucleotide pyrophosphatase (NPP) ectoenzymes also will be molecularly identified in cell lines, and their relative function studied after targeted inhibition of expression by RNA interference. A key component in these analyses will be quantification of ATP at the cell surface using a cell-attached luciferase-based assay. Accomplishment of the goals of this research will provide new molecular insight into the biology of nucleotide-stimulated P2Y receptor signaling and will provide much-needed new pharmacological agents to selectively activate and block each of these drug targets with high affinity.